Chronic obstructive pulmonary disease (COPD) affects 15 million patients in the U.S. and is the sixth leading cause of death. It is characterized by the retention of mucus secretions in the lungs. Many patients diagnosed with COPD have a disorder called chronic bronchitis (CB), and 600,000 patients are hospitalized each year due to an acute exacerbation of CB. Cystic fibrosis and Primary Ciliary Dyskinesia (PCD) are other examples of lung disorders which assume a clinical profile similar to COPD. Ciliary dyskinesia, whether primary or secondary, results in retained secretions that can only be cleared by coughing.
Another disease state characterized by the accumulation of retained mucous secretions is sinusitis. Sinusitis is an inflammation of the paranasal sinuses typically associated with an upper respiratory infection. It is this country's most common health-care complaint, affecting an estimated 31 million people. (A. Moss and V. Parsons, National Center for Health Statistics, 1986: 66-7, DHHS Publication No. (PHS)86-1588 (1985)).
Otitis media (OM) is a viral or bacterial infection of the middle ear which primarily afflicts children under the age of three. It is usually precipitated by an upper respiratory infection which spreads into the middle ear via the nasopharynx and eustachian tube. Approximately 25-50 million office visits are made each year for diagnosis and treatment of OM. By age three, about 75% of children will have had at least one episode of acute OM (J. Klein, Clin. Infect. Dis. 19, 823-33 (1994)). Following appropriate treatment with antibiotics, accumulated fluid in the middle ear remains, causing hearing impairment and potential language and cognitive development delays. Enhanced ability to clear secretions in the middle ear would reduce or eliminate significant sequelae of otitis media.
An additional disorder resulting from retained secretions is pneumonia. Patients who are immobilized for a variety of reasons are at high risk for developing pneumonia. Despite extra vigilance and numerous interventions, pneumonia develops in over 400,000 patients per year, with significant morbidity and mortality.
There are also situations where it is therapeutically desirable to increase drainage of the lacrimal system. When the lacrimal drainage system is not functioning properly the result can be excessive tearing (epiphora), mucopurulent discharge, and recurrent dacryocystitis. Current treatments for nasolacrimal duct obstruction are mostly invasive surgical procedures, and researchers have sought to discover noninvasive pharmaceutical treatments.
Tear secretion can be stimulated from lacrimal accessory tissues via P2Y2 and /or P2Y4 purinergic receptor-mediated mechanisms similar to those which hydrate airway epithelia. Dry eye disease is the general term for indications produced by abnormalities of the precorneal tear film characterized by a decrease in tear production or an increase in tear film evaporatioin, together with the ocular surface disease that results. Currently, the pharmaceutical treatment of dry eye disease is mostly limited to administration of artificial tears (saline solution) to temporarily rehydrate the eyes. However, relief is short lived and frequent dosing is necessary.
Normally, mucous secretions are removed via the mucociliary clearance (MCC) system. MCC relies on the integrated action of three components: 1) mucus secretion by goblet cells and submucosal glands; 2) the movement of cilia on epithelial cells which propels the mucus across the luminal surface; and 3) ion transport into and out of luminal epithelial cells which concomitantly controls the flow of water into the mucus.
It is now known that nucleoside phosphates such as uridine 5′-triphosphate (UTP) modulate all of the components of the MCC system. First, UTP has been shown to increase both the rate and total amount of mucin secretion by goblet cells in vitro (M. Lethem, et al., Am J. Respir. Cell Mol. Biol. 9, 315-22 (1993)). Second, UTP has been shown to increase cilia beat frequency in human airway epithelial cells in vitro (D. Drutz, et al., Drug Dev. Res. 37(3), 185 (1996)). And third, UTP has been shown to increase Cl− secretion, and hence, water secretion from airway epithelial cells in vitro (S. Mason, et al., Br. J. Pharmacol. 103, 1649-56 (1991)). In addition, it is thought that the release of surfactant from Type II alveolar cells in response to UTP (Gobran, Am. J. Physiol. 267, L625-L633 (1994)) contributes to optimal functioning of the lungs and may assist in maximizing MCC (M. Knowles, et al., N. Engl. J. Med. 325, 533-38 (1991)). UTP has been shown to increase intracellular Ca++ due to stimulation of phospholipase C by the P2Y2 receptor (H. Brown, et al., Mol. Pharmocol. 40, 648-55 (1991)).
UTP's modulation of all components of the mucociliary escalator system results in a 2.5-fold improvement in lung mucociliary clearance in normal volunteers without any significant side-effects (K. Olivier, et al., Am. J. Respir. Crit. Care Med. 154, 217-23 (1996)). In addition, UTP significantly enhanced cough clearance (clearance of retained secretions by coughing) in patients with PCD (P. Noone, et al., Am. J. Respir. Crit. Care Med. 153, A530 (1996)).
Because of UTP's demonstrated ability to increase the clearance of retained mucous secretions, applicants were motivated to investigate whether other nucleoside phosphates could be equally, if not more, therapeutically effective. The present invention is based upon this investigation.
Previously described dinucleotides are listed in Table I, along with their corresponding literature references.
TABLE IDINUCLEOTIDES IN THE LITERATURE(numbers in parentheses correspond to references that follow)Np2NNp2N′Np3NNp3N′Np4NNp4N′Ap2A (4, 1)Ap2NAD (6)Up3U (1)Ap3T (20)Up4U (2, 3)Ap4U (3)Gp2G (5, 1)Ap2TAD (6)Ap3A (1, 4, 29)m7Gp3G (5)Ap4A (1, 4, 29)Ap4C (3)m7Gp2m7G (5)Ap2C-NAD (6)Xp3X (1)m2,2,7Gp3G (5)Cp4C (3)Ap4G (3)Ap2C-PAD (6)m7Gp3m7G (5)m2,7Gp3G (5)Gp4G (1, 5)Gp4U (3)Ap2BAD (6)Gp3G (1)Xp4X (1)Gp4C (3)m7Gp2G (5)Dp4D (15)Up4C (3)Up2U (43)eAp4eA (7)Ap4T (20)m7Gp4m7G (5)m7Gp4G (5)m2,7Gp4G (5)m2,2,7Gp4G (5)Np5NNp5N′Np6NNp6N′Np8NAp5A (4)Ap5T (20)Ap6A (4)Ap6T (20)Ap8A (4)AppZppADppZppDApZppZpAApSpZpSpAZZZZCH2 (8)CH2 (15)CH2 (8)CHF (8)CH2CH2 (8)CH2CH2 (15)CH2CH2 (8)CF2 (8)CHF (8)CHF (15)CHF (8)O (8)CF2 (8)CF2 (15)CF2 (8)CHCl (8)CHCl (15)CHCl (8)CCl2 (8)CCl2 (15)CCl2 (8)A = Adenosine U = Uridine G = Guanosine T = Thymidine X = Xanthosine TAD = Tiazofurin BAD = Benzamide riboside D = 2,6-Diaminopurine eA = Ethenoadenosine m7G = 7-Methylguanosine m2,7G = 2,7-Dimethylguanosine m2,2,7G = 2,2,7-Trimethylguanosine NAD = nicotinamide riboside C-NAD = C-nicotinamide riboside C-PAD = C-picolinamide riboside N = Nucleoside (1) M. A. G. Sillero et al., Eur. J. Biochem., 76, 331 (1977) (2) C. G. Vallejo et al., Biochim. Biophys. Acta, 483, 304 (1976) (3) H. Coste et al., J. Biol. Chem., 262, 12096 (1987) (4) K. E. Ng et al., Nucleic Acid Res., 15, 3573 (1987) (5) J. Stepinski et al., Nucleosides & Nucleotides, 14, 717 (1995) (6) A. Zatorski et al., J. Med. Chem., 39, 2422 (1996) (7) P. Rotilan et al., FEBS, 280, 371 (1991) (8) P. C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992) (9) J. Walker et al., Biochemistry, 32, 14009 (1993) (10) R. H. Hiderman et al., J. Biol. Chem., 266, 6915 (1991) (11) J. Luthje et al., Eur. J. Biochem., 173, 241 (1988) (12) R. H. Silverman et al., Microbiological Rev., 43, 27 (1979) (13) C. D. Lobaton et al., Eur. J. Biochem., 50, 495 (1975) (14) G. Lowe et al., Nucleosides & Nucleotides, 10, 181 (1991) (15) G. M. Blackburn et al., Nucleosides & Nucleotides, 10, 549 (1991) (16) J. C. Baker et al., Mutation Res., 208, 87 (1988) (17) G. Klein et al., Biochemistry, 27, 1897 (1988) (18) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990) (19) D. R. Elmaleh et al., Proc. Natl. Acad. Sci., 81, 918 (1984) (20) R. Bone et al., J. Biol. Chem., 261, 16410 (1986) (21) Fed. Amer. Soc. Exper. Bio., Abstr. Part I, no. 1878 (1991) (22) M. T. Miras-Portugal et al., Ann. NY Acad. Sci., 603, 523 (1990) (23) A. Guranowski et al., Biochemistry, 27, 2959 (1988) (24) F. Grummt et al., Plant Mol. Bio., 2, 41 (1983) (25) A. G. McLennan et al., Nucleic Acid Res., 12, 1609 (1984) (26) P. Zamecnik et al., Analytical Biochem., 134, 1 (1983) (27) F. Rapaport et al., Proc. Natl. Acad. Sci., 78, 838 (1981) (28) T. Kimura et al., Biol. Pharm. Bull., 18, 1556 (1995) (29) B. Schulze-Lohoff et al., Hypertension, 26, 899 (1995) (30) B. K. Kim et al., Proc. Natl. Acad. Sci., 89, 11056 (1992) (31) P. C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992) (32) H. Morii et al., Eur. J. Biochem., 205, 979 (1992) (33) E. Castro et al., Pflugers Arch., 426, 524 (1994) (34) H. Schluter et al., Nature, 367, 186 (1994) (35) E. Castro et al., Br. J. Pharmacol., 206, 833 (1992) (36) T. Casillas et al., Biochemistry, 32, 14203 (1993) (37) J. Pintor et al., J. Neurochem., 64, 670 (1995) (38) E. Castro et al., J. Biol. Chem., 270, 5098 (1995) (39) V. A. Panchenko et al., Neuroscience, 70, 353 (1996) (40) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990) (41) J. Pintor et al., Gen. Pharmac., 26, 229 (1995) (42) J. Pintor et al., Br. J. Phamacol., 115, 895 (1995) (43) A. Kanavarioti et al., Tett. Lett., 32, 6065 (1991) 